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  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/12—Viral antigens
• • A—HUMAN NECESSITIES
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  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/12—Viral antigens
  • A61K39/23—Parvoviridae, e.g. feline panleukopenia virus
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/02—Bacterial antigens
  • A61K39/0225—Spirochetes, e.g. Treponema, Leptospira, Borrelia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/02—Bacterial antigens
  • A61K39/0241—Mollicutes, e.g. Mycoplasma, Erysipelothrix
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  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
  • A61K2039/552—Veterinary vaccine
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/70—Multivalent vaccine
• • C—CHEMISTRY; METALLURGY
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  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
  • C12N2750/00011—Details
  • C12N2750/14011—Parvoviridae
  • C12N2750/14311—Parvovirus, e.g. minute virus of mice
  • C12N2750/14334—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
• • C—CHEMISTRY; METALLURGY
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  • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
  • C12N2770/00011—Details
  • C12N2770/10011—Arteriviridae
  • C12N2770/10034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

• the invention in general pertains to the field of swine health. Swine are prone to many pathogenic micro-organisms. Control of infection is commonly done by stable and feed management, treatment with pharmaceuticals such as anti-viral drugs and antibiotics, or prophylactic treatment using vaccines.
• the invention pertains to vaccines against PRRS (porcine reproductive and respiratory syndrome) virus, Erysipelothrix rhusiopathiae, porcine parvo virus and Leptospira interrogans (Sensu Lato), and to a method of protecting an animal against such infections using such vaccines.
• PRRS progen reproductive and respiratory syndrome
• PRRS virus was first reported in 1987 in North America and Central Europe.
• PRRS virus is a small, enveloped RNA virus. It contains a single-stranded, positive-sense, RNA genome with a size of approximately 15 kilobases. The genome contains nine open reading frames.
• the virus is a member of the genus Arterivirus, family Arteriviridae, order Nidovirales.
• the two prototype strains of PRRSV are the North American strain, VR-2332, and the European strain, the Lelystad virus (LV).
• the European and North American PRRSV strains cause similar clinical symptoms. In the early 2000s a highly pathogenic strain of the North American genotype emerged in China.
• Porcilis® PRRS (available from MSD Animal Health, Boxmeer, The Netherlands) is a vaccine comprising live attenuated PRRS virus type I and is registered to reduce infection (viraemia) caused by infection with PRRS virus.
• Ingelvac PRRS® MLV (available from Boehringer
• Ingelheim, Ingelheim, Germany is a vaccine that aids in the reduction of disease caused by PRRS virus.
• Fostera® PRRS (available from Zoetis, Florham Park, New Jersey, USA) is also a MLV vaccine and is registered for protection against both the respiratory and reproductive forms of disease caused by PRRS virus.
• Other PRRS vaccines are described for example in WO2006/074986, US 8728487 and
• Erysipelothrix rhusiopathiae Infectious disease caused by Erysipelothrix rhusiopathiae (Ery) in pigs is known as erysipelas and is one of the oldest recognized diseases that affect growing and adult swine. Up to 50% of pigs in intensive swine production areas are considered to be colonized with Ery. The organism commonly resides in the tonsillar tissue. These typical healthy carriers can shed the organism in their faeces or oronasal secretions and are an important source of infection for other pigs. Disease outbreaks may be acute or chronic, and clinically inapparent infections also occur.
• Acute outbreaks are characterized by sudden and unexpected deaths, febrile episodes, painful joints, and skin lesions that vary from generalized cyanosis to the often-described diamond skin (rhomboid urticaria) lesions.
• Chronic erysipelas tends to follow acute outbreaks and is characterized by enlarged joints and lameness.
• a second form of chronic erysipelas is vegetative valvular endocarditis. Pigs with valvular lesions may exhibit few clinical signs; however, when exerted physically they may show signs of respiratory distress, lethargy, and cyanosis, and possibly suddenly succumb to the infection.
• Acutely affected pregnant sows may abort, probably due to the fever.
• Vaccination is very effective in controlling disease outbreaks.
• Injectable bacterins and other non-replicating immunogens are known and provide a long duration of immunity.
• Commercial available vaccines comprising non- replicating immunogens of Erysipelothrix rhusiopathiae are Porcilis® ERY+Parvo (MSD Animal Health), FarrowSure® Gold (Zoetis), ErySeng® Parvo (Hipra) and
• PARVORUVAX® (Merial). Optimal timing of vaccination may vary from farm to farm. Susceptible pigs may be vaccinated before weaning, at weaning, or several weeks after weaning. Male and female swine selected for addition to the breeding herd should preferably be vaccinated with a booster 3 to 5 weeks later. Thereafter, breeding stock should be vaccinated twice yearly. In general there is good cross-protection among the major Erysipelothrix rhusiopathiae strains infecting pigs. Porcine parvovirus is ubiquitous in pigs around the world. Almost all females are naturally infected before their second pregnancy, and immunity is lifelong.
• Gilts that are immunologically naive or have high passive antibody titers have the highest risk of reproductive disorders caused by the virus. Infection before day 30 of pregnancy results in early embryonic loss. Fetal infection between 30 and 70 days of gestation can result in death of the fetus and mummification. Not all fetuses are infected at the same time, and death at different stages of pregnancy is typical. Some fetuses survive and are born alive but persistently infected. Most fetuses infected after 70 days of gestation mount an immune response, clear the virus, and are healthy at birth.
• Leptospira interrogans (Sensu Lato, i.e. all pathogenic leptospira bacteria), especially serogroup Pomona, is a major cause of reproductive failure in swine (infertility, abortion, stillbirths, and the birth of weak piglets). Although acute leptospirosis occurs in adult swine, most cases are asymptomatic. Pigs infected with serogroups Pomona and Australis, serovar Bratislava, can become chronic renal carriers. Abortion occurs 1 to 4 weeks after infection, and the faetuses are autolyzed. Mummification, maceration, stillbirths, and weak pigs are also seen.
• Diagnosis is based on demonstration of leptospires in fetal tissues or stomach contents. However, severely autolyzed fetuses may result in poor fluorescent antibody and immunohistochemistry results. PCR testing has better sensitivity and specificity. Vaccination with a (multivalent) bacterin or other non-replicating immunogen, typically every 6 to 12 months, helps mitigate or even prevent leptospirosis. Field results indicate that Leptospira infection cannot be reliably eliminated with antibiotics. Effective vaccines can be obtained commercially, e.g.
• a new vaccine for the protection of swine against infections with various disease causing micro-organisms comprising in combination non-replicating immunogens of Erysipelothrix rhusiopathiae, porcine parvo virus, Leptospira interrogans and live attenuated PRRS virus, and a pharmaceutically acceptable carrier wherein the vaccine is administered in a single dose with regard to the treatment against an infection with PRRS virus.
• This vaccine is very suitable to be used in female swine for improving their reproductive performance.
• the vaccine is administered in a single dose with regard to the treatment against an infection with PRRS virus. It was advantageously found that a swine can be vaccinated successfully with the combination vaccine against PRRS virus infection even after a single shot administration of the vaccine. This does not exclude that a follow up vaccination is given, for example 6 to12 months after the first vaccination to renew the level of protection.
• This follow up vaccination differs from a boost vaccination in a prime-boost vaccination scheme, wherein protection is only believed to be obtained after the boost vaccination. In a prime-boost scheme, the two vaccinations are typically 2-4 weeks apart.
• PRRS virus is an immune evasive virus against which adequate protection is not easy to arrive at.
• a one shot vaccination is sufficient when using a live attenuated PRRS virus in combination with the Ery, paro and Lepto antigens, even in PRRS virus negative animals. This is not understood, but may be a result of positive antigen interference.
• the present invention also pertains to a combination vaccine for use in a method for prophylactic treatment of a swine against an infection with Erysipelothrix rhusiopathiae, porcine parvo virus, Leptospira interrogans and PRRS virus, to a method to constitute such a vaccine and to a method for prophylactic treatment of a swine against an infection with Erysipelothrix rhusiopathiae, porcine parvo virus, Leptospira interrogans and PRRS virus, comprising administering the said vaccine to the animal, in particular a single dose with regard to PRRS virus,
• parenterally in particular intramuscularly.
• the invention also pertains to a combination of a first vaccine comprising non-replicating immunogens of Erysipelothrix rhusiopathiae, porcine parvo virus and Leptospira interrogans, and a pharmaceutically acceptable carrier, and a second vaccine comprising freeze-dried live attenuated PRRS virus, and an instruction that the second vaccine can be mixed with the first vaccine to form the vaccine according to the invention.
• the first and second vaccine may be sold as separate, stand-alone vaccines, but having an indication on the label, package leaflet or otherwise, that the two vaccines can be mixed to form one multi-way combination vaccine, wherein the vaccine is administered in a single dose with regard to the treatment against an infection with PRRS virus.
• a vaccine is a pharmaceutical composition that is safe to administer to a subject animal, and is able to induce protective immunity in that animal against a pathogenic microorganism (a "pathogen"), i.e. to induce a successful prophylactic treatment against an infection with the pathogen as defined here below.
• a vaccine may be used in conjunction with an adjuvant, i.e. a substance or composition that is able to increase the immune response induced by the vaccine.
• Non-replicating immunogen of a pathogen is any substance or compound
• non-replicating immunogens are inactivated (killed) whole pathogens and subunits of these pathogens such as capsid proteins and surface expressed proteins, for example recombinantly expressed proteins.
• Non-replicating immunogens e.g. killed whole pathogen, cell lysate, subunit, etc.
• evoke an immune response that is primarily of the humoral type (i.e. induction of antibodies).
• Prophylactic treatment against an infection with a pathogen is aiding in preventing or ameliorating an infection with that pathogen or a disorder arising from that infection, resulting from a post treatment challenge with a pathogen, in particular to reduce its load in the host after such challenge and optionally to aid in preventing or ameliorating one or more clinical manifestations resulting from the post treatment infection with the pathogen.
• a live attenuated pathogen is a viable, replication competent form of the pathogen having reduced virulence.
• the process of attenuation takes an infectious pathogen and alters it so that it becomes harmless or less virulent, typically by either multiple passages of the pathogen through cell systems or by genetically modifying the pathogen.
• Single dose administration of a vaccine for use in prophylactic treatment means that in order to arrive at protective immunity, the vaccination does not need to be boosted with a second administration of the vaccine.
• the first (prime) vaccination is typically boosted within 6 weeks from the first administration, commonly within 3 or even 2 weeks from the first administration, and only after the second (boost) administration protective immunity, i.e. a successful prophylactic treatment as defined here above, may be obtained.
• a pharmaceutically acceptable carrier is a biocompatible medium, viz. a medium that after administration does not induce significant adverse reactions in the subject animal, capable of presenting the antigen to the immune system of the host animal after administration of the vaccine.
• a carrier can be a liquid containing water and/or any other biocompatible solvent, possibly forming an emulsion with one or more
• hydrophobic liquids such as an oil.
• the carrier however can also be a solid such as commonly used to obtain freeze-dried vaccines (based on sugars and/or proteins).
• the non-replicating immunogens of Erysipelothrix rhusiopathiae, porcine parvo virus, Leptospira interrogans are inactivated pathogens of Erysipelothrix rhusiopathiae, porcine parvo virus and Leptospira interrogans respectively.
• a simple way is provided to have (all) immunogens available in a non-replicating form.
• any subunit vaccine might also be suitable for use in the present vaccine, by having the inactivated pathogens available, the relevant immunogens are present per se, and in a way (similar to the way wherein) they are present in the live, naturally occurring pathogen.
• the Leptospira interrogans pathogen comprises bacteria of the serogroup Pomona, which is the most important swine leptospira pathogen.
• bacteria of at least one the serogroups Tarassovi, Australis, Grippotyphosa,
• the bacteria of the serogroup Australis in particular are bacteria of the serovar Bratislava.
• the vaccine for use in a method for prophylactic treatment of a swine against an infection with Erysipelothrix rhusiopathiae, porcine parvo virus, Leptospira interrogans and PRRS virus, the non- replicating immunogens of Erysipelothrix rhusiopathiae, porcine parvo virus, Leptospira interrogans are inactivated (e.g. killed) pathogens.
• the Leptospira interrogans pathogen comprises bacteria of the serogroup Pomona, and optionally bacteria of at least one the serogroups Tarassovi, Australis, Grippotyphosa,
• the vaccine is administered parenterally, i.e. administered elsewhere to the body than the mouth and alimentary canal.
• the vaccine can for example be administered intramuscularly.
• the invention also pertains to a method to constitute the combination vaccine wherein the method comprises mixing a first composition comprising live attenuated PRRS virus with a second composition comprising the immunogens of Erysipelothrix rhusiopathiae, porcine parvo virus, and Leptospira interrogans in the pharmaceutically acceptable carrier.
• the first composition comprises freeze-dried PRRS virus, for example in a stabiliser such as known from Porcilis® PRRS.
• the mixing takes preferably place at most 24 hours before the vaccine is administered to the animal, or at most 12, 1 1 1 , 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 , or 1 ⁇ 2 an hour before administration is due to take place.
• the study was designed as a historically controlled field study. It was conducted on a farm with a history of increased abortion rates, associated with leptospira infection. High antibody titres against serogroup Pomona were found in a number of animals indicating a recent Leptospira serogroup Pomona infection. The farm was negative for PRRS virus infection.
• This vaccine composition is denoted as "Ery/parvo/lepto".
• the animals were re-vaccinated with Ery/parvo/lepto in the second week of every following lactation. New replacement gilts were vaccinated using the same schedule.
• the reproductive performance of the pigs in the study was monitored.
• the farrowing results and relevant breeding data were collected to determine if vaccination had any effect on the incidence of abortion.
• the results obtained during the study period were compared to the historical data. Since parvovirus and Ery also have a significant negative impact on reproduction (parvo may kill the foetuses and Ery may cause abortion), a substantial improvement in this respect is a good indication that the vaccine is also effective against these pathogens.
• the objective of this study was to evaluate the vaccine potential in six-week-old SPF piglets of a live attenuated Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Type 1 vaccine (Porcilis PRRS) reconstitution in a vaccine comprising in combination non replicating immunogens of Erysipelothrix rhusiopathiae, porcine parvo virus and Leptospira interrogans.
• PRRSV Porcine Reproductive and Respiratory Syndrome Virus
• Type 1 vaccine Porcilis PRRS
• the study was performed with fourty PRRSV antibody negative piglets, evenly distributed over 2 groups.
• Groups 1 and 2 were vaccinated once intramuscularly (IM) with 2 ml Porcilis PRRS diluted to 4.5log-i 0 TCID 50 per dose (this dose represents a dose slightly above minimal dose of 4.0log-i 0 TCID 50 for Porcilis® PRRS).
• Group 1 received the PRRS vaccine diluted in an experimental Ery/Parvo/Lepto combination vaccine as indicated here above in experiment 1.
• the vaccines were mixed at a temperature of 25°C and left for at least 1 hour before actual administration to the animals.
• Group 2 received the live attenuated virus diluted in Diluvac Forte, and thus de facto received the regular vaccine Porcilis® PRRS slightly above its minimal dose. This group in fact served as a positive control.
• immunogens of Erysipelothrix rhusiopathiae and live attenuated PRRS virus will also be safe and effective.
• non-replicating immunogens of porcine parvo virus may be added to provide also protection against an infection with porcine parvo virus.
• Such a three-way vaccine is also understood to be safe and effective based on the present results.
• a basic vaccination for example a prime- boost vaccination, of an animal before its first gestation (typically between 20 and 30 weeks of age) with a combination vaccine comprising non-replicating immunogens of Erysipelothrix rhusiopathiae, porcine parvo virus and Leptospira interrogans, wherein, if a prime-boost scheme is used, either the prime or boost vaccine is combined with live attenuated PRRS virus in line with the present invention (the PRRS component of the vaccine in any case being used as a single dose vaccine).
• a prime-boost vaccination could for example take place at 20 and 24 weeks age. This basic vaccination could then be repeated yearly against ery/parvo/lepto/prrs infection with a single dose of the combination vaccine according to the present invention, or with a combination vaccine wherein, depending on the circumstances, one or more of the antigens are not present (for example no leptospira antigens if a farm is not expected to be infected with any leptospira bacteria).
• PRRS depending on the PRRS infection pressure on the farm, vaccination could be repeated every 6 months (or even every 3 months) if deemed necessary.
• Such a re-vaccination at a shorter interval could be done with a combination vaccine comprising next to the live attenuated PRRS virus, non-replicating immunogens of Erysipelothrix rhusiopathiae.
• a combination vaccine comprising next to the live attenuated PRRS virus, non-replicating immunogens of Erysipelothrix rhusiopathiae.
• leptospira and parvo virus such a short term re-vaccination is typically not regarded as necessary since leptospira and parvo vaccines may have a duration of immunity of about a year in swine.
• Ery+Parvo+Lepto (EPL) consists of two vaccinations with a 4-week interval.
• Porcilis® PRRS can be co-administered during EPL primary vaccination or during the EPL booster vaccination. Both mixing options have been tested in this study.
• Group 1 was vaccinated with Porcilis EPL + Porcilis PRRS at 20w of age and with Porcilis EPL at 24w of age.
• Group 2 was vaccinated with Porcilis EPL at 20w of age and with Porcilis EPL + Porcilis PRRS at 24w of age.
• Group 3 was vaccinated with Porcilis EPL at 20w of age and at 24w of age.
• Group 4 was vaccinated with Porcilis PRRS at 20w of age. During one 20w after vaccination blood was sampled regularly and the sera were tested in the respective serological tests.
• the test was valid if the animals are seronegative at the start of the study and if at least 10 animals in each control group remains sero-negative for the component omitted from the vaccinations i.e. group 4 negative for Ery, parvo and Leptospira and group 3 negative for PRRS. These criteria were fulfilled.
• the overall results per antigen are described here below.
• the HI test is often used to detect post-infection Parvo antibodies it is less suitable for post-vaccination serological studies because after vaccination many non- responders are found. Therefore, this antigen was tested in the HI test as well as in a more sensitive commercially available ELISA.
• the MAT (micro agglutination) test is generally used to detect post-infection Leptospira antibodies. However, this test mainly measures IgM antibodies which are shortly lived and predominantly induced after primary vaccination, whereas mainly IgG antibodies are induced after a booster vaccination. In addition, some serotypes do hardly respond in the MAT test which also makes this test less suitable for serological studies. Because IgG has been implicated in protection and because the MAT test is less suitable, serotype specific antibody ELISA's were in-house developed and validated. These serotype specific inhibition ELISA's were used to measure serotype specific IgG responses in this study.
• the objective of this experiment was to evaluate the efficacy of a live attenuated PRRSV Type 2 vaccine (Prime Pac® PRRS) reconstituted at the minimal dose of 4.0 Iog10 TCID50/animal in an EPL vaccine.
• piglets seronegative for PRRSV Sixty-six 5-week-old piglets seronegative for PRRSV were included in this study.
• the piglets were vaccinated with a minimal dose of Prime Pac® PRRS reconstituted in either an EPL formulation (group 1 ; one hour of waiting time at 25°C between reconstitution and vaccination) or in Diluvac Forte (group 2), intramuscularly in the right side of the neck.
• Piglets in group 3 were vaccinated intramuscularly in the right side of the neck with 2 ml of the same EPL formulation and served as PRRSV-challenge controls.
• the piglets were challenged with a dose of 5.0 Iog10 TCID50 of a virulent PRRSV Type 2 strain, Iowa-1 , by the intranasal (IN) route, 1 ml per nostril.
• Blood samples from the piglets were taken at day of vaccination, day of challenge, 5, 7, 10, 14 and 28 days post challenge. Rectal temperatures were measured on 1 day before challenge, just before challenge, 4 hours post challenge, and thereafter daily from 1 until 10 days post challenge.
• Bodyweight were measured on the day before challenge, 9 and 25 days post challenge. On 10 days post challenge 1 1 pigs per group were euthanized and observed for lung lesions. On 28 days post challenge the remaining piglets were euthanized. Body temperature and body weight
• the mean virus titer per group is represented in table 4.
• the table clearly shows that the vaccinated animals (group 1 and 2) have on average a reduced virus load in their serum as compared to the control animals (group 3). There is little difference in height of the viremia between each of the vaccinated groups.

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